NADH dehydrogenase and succinate dehydrogenase are the primary entry points of electron equivalents into the mitochondrial respiratory chain. The enzymes are flavoproteins containing iron-sulfur centers. They have been obtained in pure and homogeneous form, and their composition determined. The aims of this project are the following: 1) Purify the subunits of NADH dehydrogenase and succinate dehydrogenase from beef heart mitochondria and determine their complete primary structures. 2) Study the active centers of both enzymes by protein chemical modifications with group specific reagents following the inactivation of the catalytic activity under different conditions of pH, buffer ionic strength, presence of substrate or competitive inhibitors. 3) Determine the substrate binding site of each enzyme by specific alkylation or affinity labelling with a radioactive substrate analog, followed by purification of the subunits and isolation of the labelled peptide. 4) Raise monospecific antibodies against the holoenzymes and the purified subunits and study their effects on the enzymic activities of the soluble and membrane bound enzymes. 5) Determine the orientation of the enzymes and the spatial arrangements of the subunits in the inner mitochondrial membrane by labelling the cytoplasmic or matrix side of the membrane with a non-penetrating radioactive probe, followed by immunoprecipitation and resolution of the labelled components by polyacrylamide gel electrophoresis in sodium dodecyl-sulfate.